Protein-DNA selectivity is a central event in many biological processes, ranging from transcription and replication to restriction and modification. Because of the excess of non-specific sequences in a cell the initial encounter between a protein and DNA usually occurs outside of the target sequence. Restriction enzymes are paradigms for the study of protein-DNA selectivity and the intermediates en route to a target sequence. We propose three aims of broad biological significance in understanding the mechanisms for targeting hydrolysis at a specific DNA site. An understanding of sequence specific cleavage is relevant to proteins mediating site specific recombination and DNA repair, and for more effective use of restriction enzymes in basic and clinical applications. 1) We will determine the structures of a BamHI isoschizomer, OkrAI, with and without DNA. OkrAI and BamHI offer an opportunity to compare two enzymes that recognize and cleave exactly the same DNA substrate, even though they differ significantly in secondary structure. 2) Specific versus non-specific DNA binding. We have determined structures of BamHI and BstYI bound to the same non-cognate DNA sequence. We will now determine the structures of BstYI bound to other non-specific DNA sequences, and image BamHI molecules diffusing along DNA. In the presence of certain co-solvents, most restriction enzymes show enhanced cleavage at non-cognate sites, the so-called "star" activity. We will determine the structures of a novel BamHI mutant that maintains fidelity under star conditions. The mutant provides an opportunity to gain a deeper understanding of the structural changes accompanying a switch between non-cognate and cognate DNA binding. 3) We will obtain structural information on EcoP15I, a prototype of ATP-dependent type III family of restriction enzymes. Despite over 30 years of biochemical and genetic studies, there is still no structural information on these multi-subunit enzymes (MW >350kDa) that consume ATP to acquire directionality on the DNA. We will put a crude understanding of this unique multi-functional ATP-dependent enzyme onto a more firm structural framework. The studies on EcoP15I, BamHI and BstYI complement each other to provide a molecular perspective on active versus passive diffusion in DNA metabolism. PUBLIC HEALTH RELEVANCE: Site-specific hydrolysis of DNA is common to many biological processes. The discovery of restriction enzymes opened the doors of modern biotechnology and they are ideal systems for the study of site-specific hydrolysis. We will uncover the extraordinary mechanisms by which these enzymes target and hydrolyze specific DNA sequences.